Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on May 19, 2026 has been entered.
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on May 19, 2026. Claims 1-5, 9-10, 12, 14-17, 21-22 and 24-27 are pending. Claims 9-10, 15-17, 21 and 27 are withdrawn. Claims 1-5, 12, 14, 22 and 24-26 are currently examined.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
(Previous Rejection – Withdrawn) Claims 1, 3-5 and 12-13 were rejected under 35 U.S.C. 102(a)(1) as being anticipated by Shim et al. (ACS Nano, 2013, 7: 5955–5964).
This rejection is withdrawn in view of the amendments filed on May 19, 2026, and in favor of the new rejections below. Applicant’s arguments regarding withdrawn the rejection are moot.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102 of this title, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
(Previous Rejection – Withdrawn) Claims 2, 14, 22 and 24-26 were rejected under 35 U.S.C. 103 as being unpatentable over Shim et al. (ACS Nano, 2013, 7: 5955–5964).
(Previous Rejection – Withdrawn) Claims 1-5, 12-14, 22 and 24-26 were rejected under 35 U.S.C. 103 as being unpatentable over Shim et al. (ACS Nano, 2013, 7: 5955–5964), as applied in the 102 rejection above, in view of Farka et al. (Angew. Chem. Int. Ed. 2020, 59, 10746–10773. First published: 23 December 2019; submitted in IDS filed on Nov. 15, 2023).
The above rejections are withdrawn in view of the amendments filed on May 19, 2026, and in favor of the new rejections below. Applicant’s arguments regarding the withdrawn rejections are moot.
(New Rejection) Claims 1-5, 12, 14, 22 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Shim et al. (ACS Nano, 2013, 7: 5955–5964), of record in the previous Office action, in view of Zuba-Surma et al. (FOLIA HISTOCHEMICA ET CYTOBIOLOGICA, Vol. 45, No. 4, 2007, pp. 279-290).
Base claim 1 is directed to a method for determining the presence of a first molecule in a sample, wherein said first molecule has specific binding affinity to a second molecule, the method comprising the steps of:
a. labeling molecules of a sample suspect of comprising said first molecule with a first labeling agent;
b. contacting said sample comprising said labeled molecules from step (a) with a second molecule labeled with a second labeling agent; and
c. determining colocalization of said first labeling agent and said second labeling agent in at least two time points, wherein said colocalization is determined when said first and second labeled molecules are flowing through a microfluidic channel,
wherein colocalization of said first labeling agent and said second labeling agent in said at least two time points is indicative of the presence of said first molecule having specific binding affinity to said second molecule in said sample, thereby determining the presence of the first molecule in the sample.
Shim teaches a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3x106 per second. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. The prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. See Abstract.
Shim teaches that the ability to sensitively detect β-galactosidase paves the way for ultrasensitive diagnostics using a bead-based ELISA to quantify very low concentrations (0.046 - 4.6 pM) of the biomarker prostate-specific antigen (PSA, molecular weight 30 kDa), reported by a single enzyme. A monoclonal antibody to the target protein was covalently coupled to 1 μm polystyrene beads to enable capture in PBS buffer and subsequent detection of PSA in a sandwich complex containing a detector antibody specifically bound to a β-galactosidase reporter (Figure 3a). The capture beads exhibited red autofluorescence after covalent functionalization with monoclonal antibody, possibly due to the intrinsic fluorescence of immunoglobin. This made it possible to count the number of beads by fluorescence imaging more easily than by using bright-field illumination, without interfering with the signal arising from enzyme-catalyzed hydrolysis of the substrate in the green part of the spectrum (Figure 4a). See paragraph spanning pages 5959-5960 and Figure 3 and 4.
Accordingly, Shim teaches a method for determining the presence of a first molecule (i.e., PSA) in a sample, wherein the first molecule, PSA, has specific binding affinity to a second molecule (i.e., biotinylated detection antibody, see Figure 3), the method comprising: (a) labeling the molecules of the sample, including PSA, with a first labeling agent (i.e., capture-antibody-coated bead); (b) contacting said sample comprising said labeled molecules from (a) (i.e., PSA captured on beads, see Figure 3a) with a second molecule labeled with a second labeling agent (i.e., the detection antibody as the second molecule and the conjugated enzyme as the second labeling agent); (c) determining, in a microfluidic droplet-based approach, the temporal localization of the first and second labeling agents (see e.g., Figure 2C); and wherein colocalization of said first labeling agent and said second labeling agent in at least two time points is indicative of the presence of said first molecule having specific binding affinity to said second molecule in said sample, thereby determining the presence of the first molecule in the sample.
However, Shim does teach that temporal localization of the first and second labeling agents is determined “continuously, under flow conditions”, as instantly claimed, even though the entire process of Shim is performed in a microfluidic device. Instead, Shim teaches that once single enzyme molecules and the fluorogenic substrate have been encapsulated, it takes a few minutes to accumulate a measurable amount of fluorescent product by a typical reporter enzyme (β-galactosidase); that an area of 2 mm x 7 mm (length x width) was therefore integrated into the microfluidic device to store femtodroplets while the enzymatic reaction occurs (Figure 1b); that this storage area is divided into 40 traps (300 x 300 μm), isolated by monolithic microfluidic valves (Figure 1b-d); that trapping the femtodroplets in this way allows the activity of specific enzymes to be monitored continuously inside thousands of droplets simultaneously (Figure 2b); and that an embedded microfluidic valve is used to flush stored droplets out of the traps and reload freshly generated femtodroplets by application and release of external pressure (about 50 psi, Supporting Movie 3). See para bridging pages 5956-5957. These teachings suggest that a storing/trapping feature is introduced in the microfluidic device to pause the flow of the sample to allow sufficient time for the enzyme to produce a measurable amount of fluorescent product for detection, but, at the same time, the design rely on inclusion of valves in the microfluidic device, which is expected to complicate the manufacture and performance of the microfluidic device. One of skill in the art would have a motivation to simplify the method of Shim by removing the storing/trapping feature included in the microfluidic device, as long as there is a way to detect the enzyme-produced fluorescent product without the storing/trap feature.
Indeed, Zuba-Surma reviews about the ImageStream system a novel tool for multiparameter cell analysis in flow. The instrument integrates the features of flow cytometry and fluorescence microscopy combined with a modern methodology for image analysis. Similar to flow cytometry, ImageStream allows analysis of a large number of cells based on their fluorescence features and provides statistical analysis of these features. See Abstract. Zuba-Surma teaches that the ImageStream system can detect fluorescence from cells in flow. See Fig. 1. It teaches that the ImageStream instrument collects images continuously acquiring a signal of 10 milliseconds duration per object on average as a result of TDI and resulting in about at approximately 3 megabytes per second of data accumulation, and that this allows the detection of low fluorescence intensity signals even when the cell image is acquired at high speed. See page 281, para bridging left and right columns.
Accordingly, teachings of Zuba-Surma indicate that the technology of obtaining continuous microscopic images of small particles (cells) in a sample under flow conditions with high detection sensitivity is available at the time of invention.
Based on the discussions above, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to combine the teachings of Shim and Zuba-Surma to arrive at the invention as claimed. One would have been motivated to do so to simplify the microfluidic device of Shim by removing the storing/trap feature of the microfluidic device. Omission of an Element and Its Function Is Obvious if the Function of the Element Is Not Desired. See MPEP 2144.4 IIA. Here, the function of the traps in the microfluidic is obvious (e.g., increasing the length of time for the sample to stay in the microfluidic device so that the enzyme can produce a measurable amount of fluorescent product for detection) and this function is not desired if there exists an imaging technology that can detect fluorescent signals continuously under flow condition. E.g., one of skill in the art would readily expect that a measurable amount of the enzyme-produced fluorescent product may be achieved by various ways for it to be detected “continuously, under flow conditions” without storing/trapping in traps built into a microfluidic device, as taught in Shim, such as by reducing the overall sample flow rate, postponing detecting time point, and/or using a more sensitive camera developed for taking images continuously.
Regarding claim 2, Shim and Zuba-Surma are silent on generating a three-dimensional image as claimed even though a fluorescent microscope is used in generating images (see Figures 2 and 4); regarding claim 14, Shim and Zuba-Surma are silent on the binding affinity of the biotinylated detection antibody to PSA in KD even though it teaches that the biotinylated polyclonal anti-PSA was obtained from R&D Systems (which is expected to have a commercially acceptable binding affinity); and, regarding claim 22, Shim and Zuba-Surma are silent on if the sample (PSA sample) can be from a subject, instead, it teaches that the study is for validation of a protype for the detection of cancer biomarker PSA in buffer.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to arrive at the invention as claimed from the teachings of Shim and Zuba-Surma, as well as availability of reagents and/or equipment at the time to the researchers, and research needs. E.g., for claim 2, one would have been motivated to use a three-dimensional fluorescent microscope available at the time of invention, e.g., a 3-D microscope disclosed in Wu et al. (Lab Chip, 2012, 12, 3566–3575, of record in the previous Office actions), to obtain 3-D images. (Wu et al. reviews optical imaging techniques in microfluidics and their applications. Wu is relied upon in the rejection of claim 2). And, e.g., for claim 14, one of skill in the art would have found it obvious to use a PSA-specific antibody as the biotinylated detection antibody with sufficient binding affinity (usually such antibodies have a binding KD around 1 nM), if the one used in Shim is not good enough.
For claims 22 and 26, one would have found it obvious to apply the prototype of Shim to the detection of a sample from a subject in need of detection for PSA. And claims 24-25 read on a scenario wherein a subject in need of a PSA assay is exposed or suspected of being exposed to an infectious agent (e.g., in a flu season), there is no reason that the PSA detection device of Shim should not be used in such a scenario.
Claims 1-5, 12-14, 22 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Shim et al. (ACS Nano, 2013, 7: 5955–5964) in view of Zuba-Surma et al. (FOLIA HISTOCHEMICA ET CYTOBIOLOGICA, Vol. 45, No. 4, 2007, pp. 279-290), as applied above, and further in view of Farka et al. (Angew. Chem. Int. Ed. 2020, 59, 10746–10773. First published: 23 December 2019; of record in the previous Office actions).
This rejection addresses the elected species of (1) a biomarker for an infectious disease for the first molecule.
Relevance of Shim and Zuba-Surma is set forth above. However, Shim teaches detection of a cancer biomarker PSA, instead of a biomarker for an infectious disease.
Farka review advances in optical single-molecule detection – supersensitive bioaffinity assays. It teaches that selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. See Abstract. Farka teaches, among others, that the Nie group[87] implemented a sandwich immunoassay for virus detection based on the parallel detection of red and green fluorescent NPs in a microfluidic channel under 488-nm laser excitation. The immunocomplex was detected by monitoring the coincidence of photon bursts in the red and green detection channels. In this way, wild-type and mutated respiratory syncytial viruses were quantified in parallel with an LOD of 4 X 106 plaque-forming units (PFU). See page 10757, right column, para 2.
Accordingly, teachings Farka indicate that there is a need for detection of biomarkers of infectious diseases, and that reagents (including binding agents for immunoassays) for microfluidic detection of infectious disease biomarkers are available at the time of the current invention.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to combine the teachings of Shim, Zuba-Surma and Farka to arrive at the invention as claimed. One would have been motivated to do so to apply the concept of biomarker molecule detection and the prototype approach disclosed in Shim in detection of a biomarker for an infectious disease, such as detection viral antigens taught in Farka. There is a reasonable expectation of success that viral proteins can be detected using the approach of Shim based on the teachings of Shim, Zuba-Surma and Farka.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NIANXIANG (NICK) ZOU whose telephone number is (571)272-2850. The examiner can normally be reached on Monday - Friday, 8:30 am - 5:00 pm, EST. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, MICHAEL ALLEN, on (571) 270-3497, can be reached. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NIANXIANG ZOU/
Primary Examiner, Art Unit 1671
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